Merkel cell carcinoma virus (MCV) is a recently-discovered member of the polyomavirus family that is associated with an aggressive form of skin cancer. Our interest in this virus stems from the fact that the sequence of the viral origin of replication is significantly different from that of better studied family members such as murine polyomavirus and simian virus 40 (SV40). All of these viruses contain multiple copies of the pentameric DNA sequence GAGGC at the origin, but the MCV origin contains both more copies and more closely spaced copies than the better studied viruses. In polyomaviruses, a single protein, large T-antigen, is all that is required for origin recognition, DNA melting and the recruitment of host cell proteins that initiate replication of the viral genome. Previously we have determined crystal structures of the T-antigen domain that binds the GAGGC sequence from SV40. Based on these structures we have generated preliminary models of the T-antigen-bound MCV origin. These models suggest significant interaction between the T-antigen domains when bound to DNA at the MCV origin, but such interactions were not observed in structures from the SV40 system. The proposed work aims to determine if the predicted interactions take place and to characterize the structures of the T-antigen-DNA complexes formed at the MCV origin. Since the N- and C- terminal regions of T-antigen are not required for in vitro replication of related viruses, we will concentrate on the MCV origin- binding domain and the helicase domain. The proposed work utilizes well established methods including DNA footprinting, gel shifts, and macromolecular crystallography. We anticipate that these studies will both reveal important, general features of viral replication and provide a structural platform for future efforts aimed at inhibiting replication of MCV. PUBLIC HEALTH RELEVANCE: Merkel cell carcinoma virus (MCV) is one of five cancer-related DNA tumor viruses that infect humans. The proposed project aims to understand how the MCV protein, T-antigen, interacts with the viral DNA so as to begin the process of copying the viral genome. We anticipate that understanding this process will lead to therapeutics that combat MCV and related viruses by blocking their ability to replicate their DNA.